Project Summary/Abstract Although many discoveries have been centered around identifying new links between inflammation and Alzheimer?s Disease (AD), the mechanisms by which cholesterol storage can alter neuro-inflammatory pathways in the AD brain is not well understood. The long term goal is to delineate the role that acyl- CoA:cholesterol acyltransferase 1 (ACAT1), the cholesterol storage enzyme, plays in the progression of AD. The overall objectives of this application are to 1) determine if ACAT1 inhibition protects against inflammation in microglia cells; and to 2) determine if knocking out myeloid ACAT1 prevents Amyloid-Beta (A?) induced neuroinflammation in mouse brains. The central hypothesis of this Supplement proposal is that ACAT1 inhibition will result in a reduction of A? induced pro-inflammatory response in microglia. The rationale for this project is based on previous work published from this lab, which demonstrates that myeloid ACAT1 blockage results in a less pro-inflammatory phenotype in both atherosclerosis and obesity mouse models. Previously this lab has shown that genetic knockout as well as delivery of microRNA targeting ACAT1 prevents Alzheimer?s Disease pathology in the triple transgenic mouse model for AD. However, the effectiveness of blocking ACAT1 to combat neuroinflammation in AD has yet to be explored. We propose to test the central hypothesis by the examining the efficacy of ACAT1 inhibition in preventing A? induced inflammation in microglia and in brain tissues that are affected by A? microinjection. To accomplish this aim immortalized and freshly isolated primary microglia cells will be treated with A? (with and without the addition of the ACAT1 specific inhibitor K604) and then evaluated for pro-inflammatory markers, by using RT-PCR, ELISA, and confocal microscopy methods. To investigate the impact of myeloid depletion of ACAT1 in vivo, A? will be delivered to mouse brains by microinjection. ELISA and immunohistochemical analysis of inflammation will be evaluated in mouse brains collected from wild type and myeloid specific ACAT1 knockout mice (Acat1-M/-M). The research proposed in this application is innovative because it will likely shed light on the uncharacterized role of myeloid ACAT1 in the neuroinflammatory processes which govern AD. This work is significant because it will further substantiate myeloid ACAT1 inhibition as an effective method to alleviate AD pathology.